NDLI: Direct detection of DNA methylation during single-molecule, real-time sequencing

Content Provider	PubMed Central
Author	Flusberg, Benjamin A. Webster, Dale Lee, Jessa Travers, Kevin Olivares, Eric Clark, Tyson A. Jonas, Korlach Turner, Stephen W.
Abstract	We describe the direct detection of DNA methylation, without bisulfite conversion, through single-molecule real-time (SMRT) sequencing. In SMRT sequencing, DNA polymerases catalyze the incorporation of fluorescently labeled nucleotides into complementary nucleic acid strands. The arrival times and durations of the resulting fluorescence pulses yield information about polymerase kinetics and allow direct detection of modified nucleotides in the DNA template, including N6-methyladenosine, 5-methylcytosine, and 5-hydroxymethylcytosine. Measurement of polymerase kinetics is an intrinsic part of SMRT sequencing and does not adversely affect determination of the primary DNA sequence. The various modifications affect polymerase kinetics differently, allowing discrimination between them. We utilize these kinetic signatures to identify adenosine methylation in genomic samples and show that, in combination with circular consensus sequencing, they can enable single-molecule identification of epigenetic modifications with base-pair resolution. This method is amenable to long read lengths and will likely enable mapping of methylation patterns within even highly repetitive genomic regions.
Related Links	http://dx.doi.org/10.1038/nmeth.1459
Ending Page	465
Page Count	5
Starting Page	461
File Format	PDF
ISSN	15487091
e-ISSN	15487105
Journal	Nature methods
Issue Number	6
Volume Number	7
Language	English
Publisher Date	2010-06-01
Access Restriction	Open
Subject Keyword	Biotechnology Cell Biology Biochemistry Molecular Biology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology Biotechnology

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
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3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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